The antibody response of mice to phosphorylcholine (PC) is under investigation with the goal of understanding why one germline-encoded antibody dominates the primary IgM response to PC while two other germline-encoded antibodies are mainly detected in secondary IgG responses. The planned experiments are aimed at discerning whether the IgM, T15-idiotype positive anti-PC response arises from one subset of B lymphocytes and the IgG, 511-idiotype positive response from a second B cell subset and, whether or not the subset restriction hypothesis is correct, discerning the role of somatic mutation in generating the observed idiotypeisotype association. The experiments further examine the role of T lymphocytes in promoting clonal growth versus differentiation to antibody secretion in PC-responsive B lymphocytes, effects which could have distinct outcomes for the accumulation of somatic mutations. Assays have been developed which detect cells secreting antibodies of each of the three idiotypes associated with the anti-PC response. These will be combined with in vitro limiting dilution analysis to generate data for the precursor frequency of B cells expressing each idiotype, the clonal burst size generated by each precursor under different conditions, and the rate of division within the clone. Clonal progeny will be captured as hybridomas by somatic cell fusion, and the mRNA of hybridoma antibodies will be sequenced to generate data on the accumulation of somatic mutations within each of the three idiotype families. The hypothesis that T15-idiotype positive anti-PC antibodies are intolerant of somatic mutation will thus be put to direct test. Important structural: functional correlates of antibody activity will emerge from this study since we will be able to compare mutations that increase antibody affinity for PC with those that decrease affinity. Additional in vitro experiments will be performed to test the activity of two distinct helper T cell lines in promoting B cell growth versus differentiation. We propose that B cells with extended clonal expansion will accumulate more somatic mutations than B cells with early differentiation to antibody secretion. These T cell lines will also be used in adoptive transfer experiments to investigate the same issues. Finally, we will fractionate B cells on the basis of the xid mutation, size, or responsiveness to anti-Lyb2 antibody to determine if any B cell subset is more or less inducible with each of the two T cell lines.